This invention relates to measuring properties of biological samples using scatterometry techniques.
Cytometry refers to the measurement of cells. These measurements can refer to a cell's physical properties (shape, volume, and so on) or of the cell's biochemical properties (protein content, lipid content, and so on). One common class of cytometry measurements is light scattering measurements, which are also referred to as scatterometry metrology. Scatterometry techniques can be used in a range of biological applications, ranging from the assessment of bacterial concentration in a suspension to the resolution of the fine structure of single cells. Scatterometry is often a preferred method for making measurements on single cells in situations where fluorometry (i.e., staining the cells with fluorophores, exciting them, and studying the scattered light) is not feasible, for example, when a population of live cells is studied and the fluorophore is toxic to the cells. Scatterometry also allows measurements to be made on any type of cells or particles, not only on cells that express a fluorescent protein, as is the case with fluorescent measurements.
When a single cell intersects a light beam, typically a laser beam, some of the light is scattered out of the beam. The amount of light that is scattered by a cell is a complex function of the cell's size, shape and refractive index. The sensitivity of a measurement to each of these factors is dependent upon the range of angles over which the scattered light is collected. For example, light scattered at small angles (i.e. forward light scatter) is most dependent upon the size of the scattering particle.
A common problem in scatterometry measurements is the existence of unwanted reflections, scatter and artifacts in the signal that is received by the detector. One such problem is illustrated in FIG. 1, which shows how light is reflected from the meniscus of a solution in a sample well and is received by the detector. FIGS. 2 and 3 show the manifestation of these meniscus reflections when the sample is imaged. The meniscus reflections can be seen as various types of large “half-moon” shaped features in the left hand side of FIGS. 2 and 3. These reflections are due to the difference in refractive index between the sample liquid and air at the meniscus interface and the shape results from the angle of the meniscus where the incoming laser light hits the meniscus, as well as the well's wall shape. Certain types of wells have reflections from the walls themselves too.
As the skilled reader realizes, these reflections are highly undesirable as they may obscure valuable scattering data originating from the sample. There is therefore a need for an improved method and apparatus for reducing specular reflections, for example, from the meniscus and improving the abilities to view the cells in the sample solution using laser scatterometry techniques.